Cytokine immunoconjugates

ABSTRACT

Immunoconjugates for the selective delivery of a cytokine to a target cell are disclosed. The immunoconjugates are comprised of an immunoglobulin heavy chain having a specificity for the target cell, such as a cancer or virus-infected cell, and a cytokine, such as lymphotoxin, tumor necrosis factor alpha, interleukin-2, or granulocyte-macrophage colony stimulating factor, joined via Aits amino terminal amino acid to the carboxy-Aterminus of the immunoglobulin. Nucleic acid sequences encoding these immunoconjugates and methods of their preparation by genetic engineering techniques are also disclosed.

[0001] This application is a continuation-in-part of copendingapplication Ser. No. 612,099, filed Nov. 9, 1990, the disclosure ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to therapies involvingthe selective destruction of cells in vivo and to compositions of matteruseful in the treatment of various cancers and viral infections. Inparticular, this invention relates to genetically engineered antibodyfusion constructs capable of targeting an infected cell, and eliciting alocalized inflammatory response such that the cell is killed orneutralized.

[0003] Tumor necrosis factor (TNF∝) and lymphotoxin (LT or TNFβ) werefirst identified on the basis of their ability to directly kill certaintumors. However, many other biological activities are now attributed tothese closely related cytokines. These include effects on a variety ofcell types, such as the induction of histocompatibility antigens andadhesion receptors, as well as those resulting in inflammation, vascularpermeability changes and mononuclear cell infiltration (Goeddel, D. V.et al. (1986) Symp. Quant. Biol. 51:597, Cold Spring Harbor; Beutler, B.and Cerami, A. (1988) Ann. Rev. Biochem. 57:505; Paul N. L. and Ruddle,N. H. (1988) Ann. Rev. Immunol. 6:407). The very short half-life of bothTNF∝ and LT ensures that these inflammatory reactions do not occursystematically, but only at the sites of release from TNF-producingcells.

[0004] This ability to elicit a localized inflammatory response could beused in the treatment of solid tumors or other diseased tissue. Forexample, if it were possible to specifically deliver either TNF∝ or LTto a tumor site, a local inflammatory response could lead to an influxof effector cells such as natural killer cells, large granularlymphocytes, and eosinophils, i.e., the cells that are needed forantibody-dependent cellular cytotoxicity (ADCC) activity.

[0005] A way to deliver the lymphokine to a specific site in vivo is toconjugate it to an immunoglobulin specific for the site. However, thefusion of protein domains to the carboxy-termini of immunoglobulinchains or fragments can have unexpected consequences for the activitiesof both the protein to be fused and the immunoglobulin, particularly asfar as antigen binding, assembly and effector functions are concerned.For example, the desired biological functions of the individual proteinsmay not be maintained in the final product.

[0006] Another potential problem with expressing proteins, such as thelymphokine LT, as a fusion protein to an immunoglobulin chain is thatthe native molecule exists in solution as a trimer and binds moreefficiently to its receptor in this form. Thus, it seems unlikely thattrimerization could still occur when LT is attached to an immunoglobulinheavy (H) chain via amino terminus and is assembled into an intact Igmolecule containing two paired H chain fusion polypeptides. Secondly,the ability of the fused LT to bind its receptor may be severelycompromised if a free amino terminus is required for receptor bindingactivity. In fact, it has been postulated that the amino andcarboxy-termini of TNF∝, and, by analogy, LT, together form a structurethat is required for receptor interaction.

[0007] It is an object of the invention to provide compositions ofmatter capable of selectively destroying cells in vivo, and therapeuticmethods for accomplishing this. It is also an object of the invention toprovide compositions of matter and therapeutic methods for selectivelydelivering a cytokine to a target cell for the purpose of destroying thetarget cell either directly or by creating an environment lethal to thetarget cell.

SUMMARY OF THE INVENTION

[0008] This invention relates to immunoconjugates which include animmunoglobulin (Ig), typically a heavy chain, and a cytokine, and to theuse of the immunoconjugates to treat disease. The immunoconjugatesretain the antigen-binding activity of the Ig and the biologicalactivity of the cytokine and can be used to specifically deliver thecytokine to the target cell.

[0009] The term “cytokinel” is used herein to describe proteins, analogsthereof, and fragments thereof which are produced and excreted by acell, and which elicit a specific response in a cell which has areceptor for that cytokine. Preferable cytokines include theinterleukins such as interleukin-2 (IL-2), hematopoietic factors such asgranulocyte-macrophage colony stimulating factor (GM-CSF), and tumornecrosis factor alpha (TNF∝).

[0010] The term “lymphokine” as used herein describes proteins, analogsthereof, and fragments thereon produced by activated lymphocytes, andhaving the ability to elicit a specific response in a cell which has areceptor for that lymphokine, e.g., lymphotoxins. Lymphokines are aparticular type of cytokine.

[0011] In preferred embodiments, the immunoconjugate comprises achimeric Ig chain having a variable region specific for a target antigenand a constant region linked through a peptide bond at the carboxyterminus of the heavy chain to the cytokine.

[0012] Immunoconjugates of the invention may be considered chimeric byvirtue of two aspects of their structure. First, the immunoconjugate ischimeric in that it includes an immunoglobulin chain (typically but notexclusively a heavy chain) of appropriate antigen binding specificityfused to a given cytokine. Second, an immunoconjugate of the inventionmay be chimeric in the sense that it includes a variable region and aconstant region which may be the constant region normally associatedwith the variable region, or a different one and thus a V/C chimera;e.g., variable and constant regions from different naturally occurringantibody molecules or from different species. Also embraced within theterm “immunoconjugate” are constructs having a binding domain comprisingframework regions and variable regions (i.e., complementaritydetermining regions) from different species, such as are disclosed byGreg Winter et al., GB2, 188, 638. Preferably, the cytokine of theimmunoconjugate can be a protein which naturally forms a dimeric ormultimeric structure when unfused, such as LT or TNF∝.

[0013] In a preferred embodiment, the chimeric Ig chain comprises aheavy (H) chain which includes the CH1, CH2 and CH3 domains. Aproteolytic cleavage site may be located between the Ig heavy chain andthe cytokine so that, when the conjugate reaches the target cell, thecytokine is cleaved from the heavy chain. A “proteolytic cleavage site”is an amino acid sequence recognizable by a protease with cleaves eitherwithin or proximal to the sequence. Preferably, the variable region isderived from a mouse (i.e. its DNA sequence or its amino acid sequenceis based on a DNA or amino acid sequence of mouse origin) and theconstant region (preferably including the framework region amino acidsof the variable region) is derived from a human; and the variable regionof the heavy chain is derived from an Ig specific for a virus-infectedcell, or for a tumor-associated or viral antigen. Preferably, thechimeric Ig chain can be assembled into the immunoconjugate by combiningit with an appropriate counterpart (light or heavy) chain to form amonovalent antigen-binding region, which can then be associated toproduce a divalent immunoconjugate specific for the target antigen.

[0014] The invention also features DNA constructs encoding theabove-described immunoconjugates, and cell lines, e.g., myelomas,transfected with these constructs.

[0015] The invention also includes a method of selectively delivering acytokine to a target cell, which method includes providing a cytokineimmunoconjugate including a chimeric Ig chain including an Ig heavychain having a variable region specific for the target cell and aconstant region joined at its carboxy terminus by a peptide bond to acytokine, and an Ig light chain combined with the chimeric Ig heavychain, forming a functional antigen-binding site, and administering theimmunoconjugate in an amount sufficient to reach the target cell to asubject harboring the target cell.

[0016] The invention thus provides an immunoconjugate in which theantigen binding specificity and activity of an antibody are combined inone molecule with the potent biological activity of a cytokine. Animmunoconjugate of the invention can be used to deliver selectively acytokine to a target cell in vivo so that the cytokine can exert alocalized biological effect, such as a local inflammatory response,stimulation of T cell growth and activation, and ADCC activity. Suchconjugates, depending on their specificity and biological activity canbe used to treat diseases involving viral infections, or cancer, bytargeted cell lysis, according to methods of the invention.

DESCRIPTION OF THE DRAWINGS

[0017] The foregoing and other objects of the present invention, and thevarious features thereof, may be more fully understood from thefollowing description, when read together with the accompanyingdrawings, in which:

[0018]FIG. 1 is a schematic representation of one embodiment of theimmunoconjugate of the present invention;

[0019]FIG. 2 is a diagram of the construction of fusion proteins betweenLT and the human Ig H chain; wherein FIG. 2A is a map of a human Cγlgene fragment cloned in plasmid pBR322; FIG. 2B shows the Cγl gene fusedto LT at the end of the CH2 domain; FIG. 2C shows the Cγl gene fused toLT at the end of the CH3 domain; FIG. 2D shows the cDNA encoding LTcloned in expression vector pDEM including promoter (arrow), the naturalleader peptide of LT (open box), the first residue of the mature protein(+1) and mouse κ L-chain poly A and 3′untranslated sequence. Open boxesrepresent protein coding regions of Cγl in A-C; black boxes representsynthetic linkers used to join the protein coding sequences; and stripedboxes represent LT coding sequences;

[0020]FIG. 3 is a photograph of an SDS-polyacrylamide gel showing ananalysis of fusion protein chain assembly, wherein chimeric ch14.18antibody is shown in lanes 1 and 4; CH2-LT is shown in lanes 2 and 5;and CH3-LT is shown in lanes 3 and 6. The position of stained markerproteins and their apparent molecular weights are indicated. The driedgel was exposed to film for either 4 hr (lanes 1 and 4) or 18 hr. Cellswere labeled with ³⁵S-methionine and secreted proteins were precipitatedwith an anti-human κ antiserum and protein A and analyzed on an SDS geleither reduced (lanes 1-3) or unreduced (lanes 4-6);

[0021]FIG. 4 is a graph showing the comparison of LT cytolyticactivities for native LT (Δ--Δ) CH2-LT (◯--◯) or CH3-LT (◯--◯, filledin) immunoconjugates. A sensitive clone of the mouse fibroblast line 929was used in the 1-day assay with mitomycin C. Relative cell survival wasquantitated by staining with crystal violet and measuring the absorbanceat 630 nm. FIG. 4A shows culture supernatants from transfected cellsassayed after first quantitating the conjugates by ELISA. FIG. 4B showspurified proteins assayed following protein A Sepharose orimmunoaffinity chromatography;

[0022]FIG. 5 is a graph of the effect of pH during purification on thecytostatic activity of CH3-LT. The activities of native LT (◯--◯),CH3-LT in culture supernatant (Δ--Δ), CH3-LT purified by protein ASepharose chromatography (¤--¤) and CH3-LT purified at pH 6.5 (Δ--Δ)were compared in the cytostatic assay (in the absence of mitomycin C)using a mouse 929 subclone;

[0023]FIG. 6 is a graph of the cytolytic and cytostatic activities of LTand CH3-LT GD2-positive M21 human melanoma cells. M21 cells were seededin 96-well plates in the [Bpresence (FIG. 6A) or absence (FIG. 6B) ofmitomycin C and dilutions of LT (◯--◯) or CH3-LT (◯--◯, filled in) wereadded. Relative cell growth was measured by staining wells with crystalviolet after 48 hr and measuring the absorbance at 630 nm;

[0024]FIG. 7 is a graph of the antigen binding activity of Ig/LTimmunoconjugates. Relative binding was determined in a competitiveantigen binding assay using ch14.18 antibody conjugated to HRP as tracerand either unlabeled ch14.18 (◯--◯), CH2-LT (◯--◯, filled in) or labeledch14.18 (¤--¤) as competitor.

[0025]FIG. 8 is a photograph of an SDS-polyacrylamide gel showing ananalysis, under reducing (R) or nonreducing (NR) conditions, of thefusion protein ch14.18-CH3-GM-CSF (lane 1) and the unfused proteinch14.18 (lane 2), where M is molecular weight markers of indicatedsizes.

[0026]FIG. 9 is a graph of GM-CSF activity of the Ig/GM-CSFimmunoconjugate ch14.18-GM-CSF (◯--◯, filled in) compared to a GM-CSFstandard (◯--◯) and conditioned medium (Δ--Δ).

[0027]FIG. 10 is a graph of TNF∝ activity of the Ig/TNFimmunoconjugatees ch14.18-TNF∝ (early) (◯--◯, filled in), ch14.18-TNF-∝(late) (Δ--Δ, filled in), compared to TNF-∝ (early) (◯--◯) and TNF-∝(late) (Δ--Δ).

DETAILED DESCRIPTION OF THE INVENTION

[0028] The invention relates to immunoconjugates useful for killing amalignant or virus-infected target cell. The immunoconjugate includes aconjugate of an antibody portion having a specificity for a surfaceantigen on a virus-infected or malignant cell, and a cytokine.

[0029]FIG. 1 shows a schematic view of a representative immunoconjugate10. In this embodiment, cytokine molecules 2 and 4 are peptide bonded tothe carboxy termini 6 and 8 of CH3 regions 10 and 12 of antibody heavychains 14 and 16. V_(L) regions 26 and 28 are shown paired with V_(H)regions 18 and 20 in a typical IgG configuration, thereby providing twoantigen binding sites 30 and 32 at the amino ends of immunoconjugate 10and two cytokine receptor-binding sites 40 and 42 at the carboxy ends ofimmunoconjugate 10. Of course, in their broader aspects, theimmunoconjugates need not be paired as illustrated.

[0030] The immunoconjugates of this invention can be produced by geneticengineering techniques; i.e., by forming a nucleic acid constructencoding the chimeric immunoconjugate. Preferably, the gene constructencoding the immunoconjugate of the invention includes, in 5′ to 3′orientation, a DNA segment which encodes a heavy chain variable region,a DNA segment encoding the heavy chain constant region, and DNA codingfor the cytokine. The fused gene is assembled in or inserted into anexpression vector for transfection of the appropriate recipient cellswhere it is expressed. The hybrid chain can be combined with a light (orheavy) chain counterpart to form monovalent and divalentimmunoconjugates.

[0031] The cytokine can be any cytokine or analog or fragment thereofwhich has a therapeutically valuable biological function. Usefulcytokines include the interleukins and hematopoietic factors such asinterleukin-2 (IL-2) and granulocyte-macrophage colony stimulatingfactor (GMCSF). Lymphokines such as LT and TNF∝, which require theformation of multimeric structures to function,-can also be used. Thegene encoding the lymphokine or cytokine can be cloned de novo, obtainedfrom an available source, or synthesized by standard DNA synthesis froma known nucleotide sequence. For example, the DNA sequence of LT isknown (see, e.g. Nedwin et al. (1985) Nucleic Acids Res. 13:6361), asare the sequences for interleukin-2 (see, e.g., Taniguchi et al. (1983)Nature 302:305-318), granulocyte-macrophage colony stimulating factor(see, e.g., Gasson et al. (1984) Science 266:1339-1342), and tumornecrosis factor alpha (see, e.g., Nedwin et al. 1. Ibid.)

[0032] The heavy chain constant region for the conjugates can beselected from any of the five isotypes: alpha, delta, epsilon, gamma ormu. Heavy chains or various subclasses (such as the IgG subclasses 1-4)can be used. The light chains can-have either a kappa or lambda constantchain. DNA sequences for these immunoglobulin regions are well known inthe art. (See, e.g., Gillies et al. (1989) J. Immunol. Meth. 125:191).

[0033] In preferred embodiments, the variable region is derived from anantibody specific for the target antigen (an antigen associated with adiseased cell such as a cancer cell or virus-infected cell), and theconstant region includes the CH1, CH2 and CH3 domains. The gene encodingthe cytokine is joined, (e.g., by appropriate linkers, e.g., by DNAencoding (Gly₄-Ser)₃ in frame to the 3′ end of the gene encoding theconstant region (e.g., CH3 exon), either directly or through anintergenic region. In certain embodiments, the intergenic region cancomprise a nucleotide sequence coding for a proteolytic cleavage site.This site, interposed between the immunoglobulin and the cytokine, canbe designed to provide for proteolytic release of the cytokine at thetarget site. For example, it is well known that plasmin and trypsincleave after lysine and arginine residues at sites that are accessibleto the proteases. Many other site-specific endoproteases and the aminoacid sequences they attack are well-known.

[0034] The nucleic acid construct can include the endogenous promoterand enhancer for the variable region-encoding gene to regulateexpression of the chimeric immunoglobulin chain. For example, thevariable region encoding genes can be obtained as DNA fragmentscomprising the leader peptide, the VJ gene (functionally rearrangedvariable (V) regions with joining (J) segment) for the light chain orVDJ gene for heavy chain, and the endogenous promoter and enhancer forthese genes. Alternatively, the gene coding for the variable region canbe obtained apart from endogenous regulatory elements and used in anexpression vector which provides these elements.

[0035] Variable region genes can be obtained by standard DNA cloningprocedures from cells that produce the desired antibody. Screening ofthe genomic library for a specific functionally rearranged variableregion can be accomplished with the use of appropriate DNA probes suchas DNA segments containing the J region DNA sequence and sequencesdownstream. Identification and confirmation of correct clones are thenachieved by DNA sequencing of the cloned genes and comparison of thesequence to the corresponding sequence of the full length, properlyspliced mRNA.

[0036] The target antigen can be a cell surface antigen of a tumor cell,a virus-infected cell or another diseased cell. Genes encodingappropriate variable regions can be obtained generally from Ig-producinglymphoid cells. For example, hybridoma cell lines producing Ig specificfor tumor associated antigens or viral antigens can be produced bystandard somatic cell hybridization techniques. (See, e.g., U.S. Pat.No. 4,96,265.) These Ig-producing cell lines provide the source ofvariable region genes in functionally rearranged form. The variableregion genes will typically be of murine origin because this murinesystem lends itself to the production of a wide variety of Igs ofdesired specificity.

[0037] The DNA fragment containing the functionally rearranged variableregion gene is linked to a DNA fragment containing the gene encoding thedesired constant region (or a portion thereof). Ig constant regions(heavy and light chain) can be obtained from antibody-producing cells bystandard gene cloning techniques. Genes for the two classes of humanlight chains and the five classes of human heavy chains have beencloned, and thus, constant regions of human origin are readily availablefrom these clones.

[0038] The fused gene encoding the hybrid IgH chain is assembled orinserted into expression vectors for incorporation into a recipientcell. The introduction of gene construct into plasmid vectors can beaccomplished by standard gene splicing procedures.

[0039] The chimeric IgH chain can be co-expressed in the same cell witha corresponding L chain so that a complete immunoglobulin can beexpressed and assembled simultaneously. For this purpose, the heavy andlight chain constructs can be placed in the same or separate vectors.

[0040] Recipient cell lines are generally lymphoid cells. The preferredrecipient cell is a myeloma (or hybridoma). Myelomas can synthesize,assemble, and secrete immunoglobulins encoded by transfected genes andthey can glycosylate protein. A particularly preferred recipient cell isthe Sp2/0 myeloma which normally does not produce endogenousimmunoglobulin. When transfected, the cell will produce only Ig encodedby the transfected gene constructs. Transfected myelomas can be grown inculture or in the peritoneum of mice where secreted immunoconjugate canbe recovered from ascites fluid. Other lymphoid cells such as Blymphocytes can be used as recipient cells.

[0041] There are several methods for transfecting lymphoid cells withvectors containing the nucleic acid constructs encoding the chimeric Igchain. A preferred way of introducing a vector into lymphoid cells is byspheroblast fusion. (see, Gillies et al. (1989) Biotechnol. 7:798-804).Alternative methods include electroporation or calcium phosphateprecipitation.

[0042] Other useful methods of producing the immunoconjugates includethe preparation of an RNA sequence encoding the construct and itstranslation in an appropriate in vivo or in vitro system.

[0043] The immunoconjugate of this invention can be used to deliverselectively a cytokine to a target cell in vivo so that the cytokine canexert a localized biological effect such as a local inflammatoryresponse, stimulation of T cell growth and activation, and ADCCactivity. A therapeutically effective amount of the immunoconjugate isadministered into the circulatory system of a subject harboring thetarget cell.

[0044] The invention is illustrated further by the followingnon-limiting Examples.

[0045] 1. Plasmid Construction

[0046] Described below is the construction of PdHL2, a plasmid whichcontains the human Cγl heavy and kappa light chain gene sequences aswell as insertion sites for V region cDNA cassettes (Gillies et al.(1989) J. Immunol. Meth. 125:191). This plasmid may be used as a starterplasmid for constructing any IgH chain cytokine fusion. For example,PdHL2 was used for the expression of Ig/LT fusion proteins. A LT cDNAwas isolated from a human peripheral blood leukocyte library cloned inλgt10. The sequence was identical to that reported in the literature byNedwin et al. (Nucleic Acids Res (1985) 13:6361). The cDNA was insertedinto vector pDEM (Gillies et al., ibid) as an XhoI fragment after firstremoving most of the 3′ untranslated region with Bal31 nuclease. Theresulting plasmid, pDEM-LT (FIG. 2), expresses (in transfected cells) afusion mRNA with a 5′ untranslated sequence derived from themetallothionein (MT) promoter, the LT coding sequence and a 3′untranslated sequence and a poly A addition signal from the mouse Cκgene. Fusion protein-encoding vectors-were constructed by ligatingHindIII to TaqI (CH2-LT) or HindIII to NsiI (CH3-LT) fragments of thehuman Cγl gene to HindIII and PvuII digested PDEM-LT using synthetic DNAlinkers (FIG. 2). These linkers:    (5′-CGAAGAAAACCATCTCCAAA/CTCCCTGGTGTTGGCCTCAC ACCTTCAG-3′ (forCH2-LT); and     5′-TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA/CTCCCTGGTGTTGGCCTCACACCTTCAG-3′)

[0047] provide the protein coding sequence from the unique site (NsiI orTaqI) to the end of the heavy-chain domain (indicated by the slash), andjoin them to the amino terminus of the mature form of LT (up to theunique PvuII site). The linker for the CH3 fusion protein also includesa silent mutation that creates a SmaI site close to the end of thedomain for future use in constructing fusion proteins. The DNA sequencesat the junction of each construct were confirmed and each HindIII toEcoRI fragment was inserted into plasmid pdHL2-VCγlκ (14.18). Thisplasmid contains the V cassettes for the ch14.18 anti-ganglioside GD2antibody (Gillies et al., ibid.).

[0048] 2. Cell Culture and Transfection

[0049] Sp2/0 Ag14 mouse hybridoma cells were maintained and transfectedas described by Gillies et al. (BioTechnology (1989) 7:8799). Drugselection in methotrexate (MTX) was initiated 24 hours aftertransfection by adding an equal volume of medium containing MTX at 0.1μM. Two additional feedings with selection medium were done at 3 dayintervals. Transfectants secreting human Ig determinants were identifiedby ELISA (Gillies et al., 1989. ibid), grown in medium containingincreasing concentrations of MTX, and subcloned by limiting dilution inmedium containing MTX at 5 μM.

[0050] 3. Purification and Characterization of Fusion Proteins

[0051] Proteins were biosynthetically labeled by incubating transfectedcells (1×10^(6/mL) for) 16 hr in growth medium containing ³⁵S-methionine(50 μCi/mL-Amersham). Culture supernatants were then clarified bycentrifugation in a microcentrifuge and the labeled proteins wereimmmunoprecipitated with polyclonal anti-human κ chain antisera (JacksonImmunoresearch, Bar Harbor, Me.) and protein A Sepharose (Repligen,Corp., Cambridge, Mass.). Protein samples were boiled for 5 min. in gelsample buffer in the presence or absence of 2-mercaptoethanol andanalyzed on a 7% polyacrylamide gel. Proteins were detected byfluorography (diphenyloxazole in DMSO) and autoradiography.

[0052] Unlabeled proteins were purified from spent suspension culturemedium by either immunoaffinity chromatography with a monoclonalanti-human κ antibody for the CH2-LT protein or by protein A Sepharosechromatography for the CH3-LT protein. All materials were concentratedby membrane dialysis into PBS. An alternative procedure for purificationof the CH3-LT protein was developed to prevent the loss of LT activityduring elution from the protein A column. Spent culture media wasdiluted with three volumes of 10 mM sodium phosphate buffer (pH 6.5) andloaded onto a Bakerbond AbX (J. T. Baker) column at room temperature.The column was washed with 10 mM sodium phosphate buffer until theabsorbance returned to baseline and then with PBS, pH 6.5 (150 mM NaCl,10 mM sodium phosphate, pH 6.5). The CH3-LT protein was eluted with 150mM NaCl, 50 mM sodium phosphate, pH 6.5.

[0053] 4. Activity Assay

[0054] The antigen binding activity of the Ig-LT proteins was measuredas described in Gillies et al. (J. Immunol. Meth. (1989) 125:191), andLT activity was determined in the cytolytic or cytostatic assay (Kahn etal. (1982)) utilizing the 159124T2.5 subclone of the mouse L929 cellline (provided by Dr. H. Schreiber, University of Chicago). Cells wereseeded into 96-well plates at 4×10⁴ cells per well, with (cytolytic) orwithout (cytostatic) mitomycin C (2 μg/mL), and 10 μL of the test samplewas added after 24 hr. Cells were stained either 24 or 48 hr later (seeFIG. descriptions) with crystal violet and the amounts of dye retainedin the wells were compared to those of untreated wells and thosereceiving the LT standard (R&D Systems). The same assay was also carriedout with the GD2-bearing human melanoma line M21, originally provided byD. L. Morton, University of California, Los Angeles. The latter cellline was also used for measuring CDC and ADCC activity as describedearlier (Gillies et al. (1990) Human Antibody. Hybridomas 1:47)

[0055] 5. Expression of Ig/LT Immunoconjugates

[0056] The Ig/LT immunoconjugates were made by directly fusing the cDNAsequence encoding the mature form of LT to the end of either the CH2 orCH3 exon of the human Cγl gene (FIG. 2) with the appropriate syntheticlinkers. This gene fusion was then combined in a vector together withthe V regions of murine antibody 14.18 and the human Cκ gene, andexpressed in transfected Sp2/0 cells. These immunoconjugates were thenexpressed and tested for antigen binding activity and Ig chain assembly.The immunoconjugates retained antigen binding when measured in acompetitive antigen binding ELISA (see below), and were assembled. Cellsexpressing these immunoconjugates were labeled with ³⁵S-methionine, andthe secreted proteins were analyzed by SDS-PAGE in the presence orabsence of reducing agent.

[0057] As seen in FIG. 3, the CH2-LT immunoconjugate was expressed as amixture of whole (approximately 180 Kd) and half (90 Kd) molecules. TheCH3-LT fusion protein, on the other hand, consisted entirely of fullyassembled molecules. This result is not surprising since the CH3 domainis most responsible for Ig chain assembly. The reason why a portion ofthe CH2-LT did assemble, i.e. formed disulfide bonds in the hinge-domainof the antibody, is likely due to the dimerization of thecarboxy-terminal LT domains.

[0058] 6. Biological Activity of Ig/LT Conjugates

[0059] The LT activities of the CH2-LT and CH3-LT conjugates werecompared in the standard cytolytic assay (Kahn, A. et al. (1982) “Astandardized automated computer assisted micro-assay for lymphotoxin.”In: Human Lymphokines, Biological response modified; (Kahn and Hill,eds.) Academic Press, New York, p. 23), using a mouse L929 subclone.This assay measures the ability of the immunoconjugate to bind to theTNF/LT receptor and trigger the active cell killing process in this cellline. When crude preparations (culture supernatants) were compared (FIG.4A), CH3-LT was found to be much more active (nearly 100 fold by thisassay) than CH2-LT and exhibited approximately the same specificactivity per mole as the LT standard. This higher activity of CH3-LT islikely due to the increased proportion of fully assembled H-chain fusionproteins. Thus, the presence of the CH3 exon in the immunoconjugate mayallow the H-chains to associate more efficiently, perhaps positioningthe LT domains in a manner that allows for dimerization and, as aconsequence, more LT receptor binding.

[0060] When purified preparations were compared, the difference inactivities between CH2-LT and CH3-LT was still evident, but the activityof the conjugates, especially CH3-LT, was greatly reduced compared tothe LT control (FIG. 4B). Since both proteins had been purified by usingelution steps at acidic pH (i.e., less than pH4), the pH sensitivity ofthe culture supernatants was examined, and the LT activity was found tobe very acid labile.

[0061] An alternative purification scheme was developed in which the pHwas not reduced to below 6.5. The material from this preparation wascompared to that purified by protein A, the original starting material,and the LT standard. The results of the LT cytostatic assay, in theabsence of mitomycin C, shown in FIG. 5, demonstrate that full LTactivity can be maintained during purification provided low pH isavoided. This assay was used to give a better dose response for the LTcontrol and to demonstrate that the relationship between CH2-LT andCH3-LT is consistent for both assay systems. The same results wereobtained in the cytolytic assay.

[0062] The results show that full activity (as measured by this assay)can be maintained when LT is fused to an Ig H chain. The fact that theLT amino terminus is covalently bound to the carboxy-terminus of theantibody apparently does not prevent LT receptor binding or the stepssubsequent to binding that are required for activating the cell killingprocess.

[0063] 7. Antigen Binding and Effector Functions of Ig/LTImmunoconjugates

[0064] The antigen binding activity of the immunoconjugates was measuredon antigen-coated plates in either a direct binding or competition assayformat. In the direct binding assay antigen binding activity was foundto be much higher than that of the control ch14.18 antibody. Since thesource of the GD2 antigen was a crude membrane extract fromneuroblastoma cells, it is possible that the TNF/LT receptor is presentin the preparation and that binding of the conjugate through the LTdomain is responsible for this increased activity. When antigen bindingwas measured in a competition assay, the conjugate was found to competewith the labeled ch14.18 antibody for antigen only slightly moreefficiently than the unlabeled ch14.18 antibody (FIG. 7).

[0065] The results show that it is possible to-combine the antigenbinding activity of an anti-tumor cell antibody with the potentbiological activity of a cytokine. The presence of the CH3 exon in theimmunoconjugate results in complete H-chain assembly and, as aconsequence, higher LT and effector activities. The assembly of H chainsmay likely result in LT dimerization.

[0066] In addition, a free amino terminus is not necessary for LTbinding to its receptor since in the highly active CH3-LTimmunoconjugate, the amino terminus of the LT domain is peptide bondedto the Ig H chain.

[0067] 8. Construction and Expression of Ig/GM-CSF Immunoconjugates

[0068] Ig/GM-CSF conjugates were made by joining a nucleotide sequenceencoding GM-CSF to a nucleotide sequence encoding an Ig heavy chain,such that the encoded protein includes a heavy chain fused via thecarboxy terminus to GM-CSF. The construct was made as follows. Themature protein coding sequence of GM-CSF was linked to the end of theCH3 exon of the human Cγ₁ gene using PdHDL2 and appropriateoligonucleotide linkers, as described above for the LT conjugate andaccording to procedures well-known in the art. Also as described abovefor LT conjugates, the Ig heavy chain GM-CSF fused gene was combinedwith the heavy chain V region gene of the 14.18 anti-GD2 heavy chain,and carried on the same vector as the human Cκ gene and the light chainV region gene of the 14.18 antibody. After transfection of the DNA intohybridoma cells and consequent expression of the H and L genes, acomplete ch14.18 antibody with GM-CSF attached to the end of each Hchain was produced. The fusion protein was purified from conditionedmedium using adsorbtion to and elution from protein A Sepharose. Thepeak material was diafiltered using an Amicon stirred cell into PBS andconcentrated to approximately 1mg/mL.

[0069] The fusion protein was analyzed by electrophoresis on a 10%SDS-polyacrylamide gel (FIG. 8) under reducing (R) or non-reducing (NR)conditions and the proteins were visualized by staining with CoomassieBlue. Lane 1, ch14.18-CH3-GMCSF; Lane 2, ch14.18; M, molecular weightmarkers of the indicated sizes in kD. The relative molecular weight ofthe fused H chain of 75 kD in lane 1 (R) is consistent with aglycosylated GM-CSF (˜25 kD) being fused to the H chain (50 kD). Note inthe non-reduced lane 1 that the fusion protein is assembled into asingle high molecular weight species of ˜200 kD.

[0070] 9. Biological Activity of Iq/GM-CSF Conjugates

[0071] The GM-CSF activity of the ch14.18-GM-CSF fusion protein wasexamined in a proliferation assay using the GM-CSF-dependent cell lineAML-193 (human acute myelogenous leukemia) (obtained from DanielSantoli, Wistar Institute, Philadelphia, Pa.). Cells are cultured for 2days in serum-free medium containing insulin and transferrin (but noGM-CSF), at which time GM-CSF or fusion protein sample dilutions areadded. After five more days, 5 μCi of ³H-thymidine is added to each welland after 16 hr, the cells are harvested in 10% trichloroacetic acid.After 30 min. on ice the precipitated material is collected on GF/Cfilters, dried and counted by liquid scintillation.

[0072] In FIG. 9, the proliferation obtained with varying amounts ofGM-CSF, conditioned medium containing the secreted fusion protein, orch14.18-GM-CSF purified by protein A Sepharose are compared. The resultsshow that significant GM-CSF activity is maintained once the molecule isfused to the H-chain but that the activity is-either 20% (conditionedmedium) or 10% that of (purified fusion protein) GM-CSF standard.Maximum incorporation was obtained with less than 10 ng/mL of thepurified fusion protein (GM-CSF equivalents or 50 ng of total protein).This slight loss of activity is not likely to affect the utility of-thisfusion protein, especially if large amounts of ch14.18-GM-CSF accumulateat the site of solid tumors expressing the GD2 antigen.

[0073] The in vivo half-life of the immunoconjugate was determined byinjecting mice (20 μg injected in the tail vein) with ch14.18-GM-CSF.Samples of blood were collected at the indicated times and the amount offusion protein in the serum was determined by ELISA. The captureantibody was a polyclonal goat anti-human IgG (Fc-specific) and thedetecting antibody was a horseradish peroxidase-conjugated goatanti-human K. As seen in Table 1, the half-life (calculated between the24 hr and 4 day time points) was nearly 3 days. This compares to thepublished value of 85 min. in humans (Herrmann et al. (1989) J. ClinOncol. 7:159-167). This increased half-life may compensate for thereduced activity of the fusion protein, especially since the localconcentration of the immunoconjugate at the tumor site is likely to beincreased by antibody targeting. TABLE 1 Serum Concentration ofch14.18-CH3-GM-CSF* Time after injection Ab Concentration (ng/mL)  4 hr9210 16 hr 9660 24 hr 5950  4 days 2530

[0074] 10. Construction, Expression, and Activity of Ig/TNFImmunoconjugates

[0075] Ig/TNF immunoconjugates were made by fusing nucleotide sequencesencoding TNFα and immunoglobulin heavy chain such that TNFα is fused tothe carboxy terminus of the heavy chain. Briefly, the mature TNFα codingsequence was fused to the end of the human Cγl CH3 exon usingoligonucleotides. The recombined fragment was joined downstream of theheavy chain V region encoding gene from the anti-GD2 mouse antibody14.18; also contained in this vector was the human κ gene, includingboth the V region gene encoding the light chain V region from theanti-GD2 mouse antibody 14.18 and the C region encoding gene. Hybridomacells were transfected and selected as described above. Clones secretinghuman antibody determinants were expanded and used for the productionand purification of the ch14.18-CH3-TNFα fusion protein by protein ASepharose chromatography. The activity of the fusion protein was testedas described above for the CH3-LT fusion proteins.

[0076] As seen in FIG. 10, the amount of cytotoxicity obtained with thefusion protein met or exceeded that of native TNF( at either early (20hr) or late (24 hr) points in the assay. This fusion protein appears tobe fully functional with respect to TNFα activity, even though it waspurified using protein A Sepharose. The CH3-LT construct was partiallyinactivated by the elution at acidic pH using the same protocol.

[0077] The results described above for the Ig/LT, Ig/GM-CSF, andIg/TNFαimmunoconjugates demonstrate that an antibody can be genetically fusedto a cytokine without the loss of antigen binding activity and effectorfunctions of the antibody, or the receptor binding and biologicalactivity of a cytokine.

[0078] 11. Dosage

[0079] Immunoconjugates of the invention may be administered at atherapeutically effective dosage within the range of lug-100mg/kg bodyweight per day. The immunoconjugate may be administered in physiologicsaline or any other biologically compatible buffered solution. Thissolution may be administered systemically (e.g., by injectionintravenously or intramuscularly).

[0080] Other Embodiments

[0081] The invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Thepresent embodiments are therefore considered to be in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

What is claimed is:
 1. A chimeric immunoglobulin (Ig) chain comprisingan Ig heavy chain and a cytokine.
 2. The chimeric Ig chain of claim 1,wherein the Ig heavy chain is joined at its carboxy-terminus by apeptide bond to the amino terminal amino acid of the cytokine.
 3. Thechimeric Ig chain of claim 1, wherein the Ig heavy chain comprises CH1,CH2, and CH3 domains.
 4. The chimeric Ig chain of claim 1, wherein aproteolytic cleavage site is located between the Ig heavy chain and thecytokine.
 5. The chimeric Ig chain of claim 1, wherein the variableregion is derived from a mouse and the constant region is derived from ahuman antibody.
 6. The chimeric Ig chain of claim 1, wherein thevariable region of the Ig heavy chain is derived from an Ig specific fora cancer cell or a virus-infected cell.
 7. The conjugate of claim 6,wherein the variable region is derived from an Ig specific for atumor-associated antigen or a viral antigen.
 8. The chimeric Ig chain ofclaim 1, wherein the cytokine is tumor necrosis factor alpha.
 9. Thechimeric Ig chain of claim 1, wherein the cytokine is interleukin-2. 10.The chimeric Ig chain of claim 1, wherein the cytokine is a lymphokine.11. The chimeric Ig chain of claim 10, wherein the lymphokine is alymphotoxin.
 12. The chimeric Ig chain of claim 10, wherein thelymphokine is granulocyte-macrophage colony stimulating factor.
 13. Thechimeric Ig chain of claim 10, wherein the lymphokine is a protein whichforms a dimeric or multimeric structure.
 14. A chimeric immunoglobulin(Ig) chain comprising an Ig heavy chain having a variable regionspecific for a target cell antigen and a heavy chain including CH1, CH2,and CH3 domains, joined, through a peptide bond, to the amino terminusamino acid of a cytokine.
 15. The chimeric Ig chain of claim 14, whereinthe cytokine is selected from the group consisting of lymphotoxin,interleukin-2, tumor necrosis factor, and granulocyte-macrophage colonystimulating factor.
 16. A cytokine immunoconjugate comprising: (a) achimeric immunoglobulin (Ig) chain including an Ig heavy chain having avariable region specific for a cancer cell or virus-infected cell,joined at the carboxy-terminus of its constant region by a peptide bondto a cytokine; and (b) an Ig light chain having a variable regionspecific for the cancer or virus-infected cell, said heavy and lightchains forming a functional antigen-binding site.
 17. Theimmunoconjugate of claim 16, wherein the chimeric heavy chain has aconstant region comprising CH1, CH2, and CH3 domains.
 18. Theimmunoconjugate of claim 16, wherein the cytokine is interleukin-2. 19.The immunoconjugate of claim 16, wherein the cytokine is tumor necrosisfactor alpha.
 20. The immunoconjugate of claim 16, wherein the cytokineis a lymphokine.
 21. The immunoconjugate of claim 20, wherein thelymphokine is lymphotoxin.
 22. The immunoconjugate of claim 20, whereinthe lymphokine is granulocyte-macrophage stimulating factor.
 23. Anucleic acid encoding a chimeric immunoglobulin (Ig) chain comprising anIg heavy chain and a cytokine.
 24. The nucleic acid of claim 23 which isDNA.
 25. The nucleic acid of claim 23, wherein the Ig heavy chaincomprises CH1, CH2, and CH3 domains.
 26. The nucleic acid of claim 23,wherein the variable region is derived from an Ig specific for a cancercell or a virus-infected cell.
 27. The nucleic acid of claim 26, whereinthe variable region is derived from an Ig specific for atumor-associated antigen or a viral antigen.
 28. The nucleic acid ofclaim 23, wherein a proteolytic cleavage site is located between the Igheavy chain and the cytokine.
 29. The nucleic acid of claim 23, whereinthe variable region is derived from a mouse antibody and the constantregion is derived from a human antibody.
 30. The nucleic acid of claim23, wherein the cytokine is interleukin-2.
 31. The nucleic acid of claim23, wherein the cytokine is tumor necrosis factor alpha.
 32. The nucleicacid of claim 23, wherein the cytokine is a lymphokine.
 33. The nucleicacid of claim 32, wherein the lymphokine is a protein which forms adimeric or multimeric structure.
 34. The nucleic acid of claim 32,wherein the lymphokine is a lymphotoxin.
 35. The nucleic acid of chain32, wherein the lymphokine is granulocyte-macrophage colony stimulatingfactor.
 36. A recombinant DNA encoding a chimeric immunoglobulin (Ig)chain, comprising an Ig heavy chain having a variable region specificfor a target cell antigen and heavy chain having CH1, CH2 and CH3domain, joined, through a peptide bond, to the amino terminal amino acidof a cytokine.
 37. The DNA construct of claim 35, wherein the cytokineis selected from the group consisting of tumor necrosis factor alpha,interleukin-2, lymphotoxin, and granulocyte-macrophage colonystimulating factor.
 38. A cell line transfected with the nucleic acid ofclaim
 23. 39. A cell line transfected with the nucleic acid of claim 36.40. A cell line of claim 23 which is a myeloma cell line.
 41. A cellline of claim 36 which is a myeloma cell line.
 42. A method ofselectively delivering a cytokine to a target cell, comprising: (a)providing a cytokine immunoconjugate including: a chimericimmunoglobulin (Ig) chain comprising an Ig heavy chain having a variableregion specific for the target cell joined at the carboxy terminus ofits constant region by a peptide bond to a cytokine, and an Ig lightchain combined with the chimeric Ig heavy chain, forming a functionalantigen-binding site; and (b) administering to a subject harboring thetarget cell an amount of the immunoconjugate sufficient to reach thetarget cell.
 43. The method of claim 42 wherein said target cell is acancer cell or a virus-infected cell.
 44. The method of claim 42,wherein the chimeric heavy chain has a constant region comprising CH1,CH2, and CH3 domains.
 45. The method of claim 42, wherein the cytokineis selected from the group consisting of lymphotoxin, interleukin-2,tumor necrosis factor alpha, and granulocyte-macrophage colonystimulating factor.